A growing family of regulatory proteins that deliver signals between cells of the immune system has been identified. Called cytokines, these proteins have been found to control the growth and development, and bioactivities, of cells of the hematopoietic and immune systems. Cytokines exhibit a wide range of biological activities with target cells from bone marrow, peripheral blood, fetal liver, and other lymphoid or hematopoietic organs. Exemplary members of the family include the colony-stimulating factors (GM-CSF, M-CSF, G-CSF, interleukin-3), the interleukins (IL-1, IL-2, IL-11), the interferons (alpha, beta and gamma), the tumor necrosis factors (alpha and beta) and erythropoietin.
Within this family of proteins, an emerging group of chemotactic cytokines, also called chemokines or intercrines, has been identified. These chemokines are basic, heparin-binding proteins that have proinflammatory and reparative activities. They are distinguished from other cytokines having proinflammatory and reparative activities (such as IL-1 and platelet-derived growth factor) by their characteristic conserved single open reading frames, typical signal sequences in the N-terminal region, AT rich sequences in their C-terminal untranslated regions, and rapidly inducible mRNA expression. See, e.g., Wolpe, FASEB J. 3:2565–73(1989) and Oppenheim, Ann. Rev. Immunol. 9:617–48(1991). Typically, the chemokines range in molecular mass from 8–10 kD; in humans, they are the products of distinct genes clustered on chromosomes 4 and 17. All chemokines have four cysteine residues, forming two disulfide bridges.
Two subfamilies of chemokines have been recognized, based on chromosomal location and the arrangement of the cysteine residues. The human genes for the α, or C-X-C, subfamily members are located on human chromosome 4. In this subfamily the first two cysteines are separated by one amino acid. The members of this subfamily, the human proteins IL-8 (interleukin-8), beta TG (beta thromboglobulin), PF-4 (platelet factor 4), IP-10, GRO (growth stimulating factor, also known as MGSF, melanoma grow stimulating factor) and murine MIP-2 (macrophage inhibitory protein-2), besides having the C-X-C arrangement of their first two cystein residues, exhibit homology in their amino acid sequences in the range of 30–50%.
In the beta subfamily, the first two cysteine residues are located adjacent to each other, a C-C arrangement. The human genes encoding the β subfamily proteins are located on chromosome 17 (their mouse counterparts are clustered on mouse chromosome 11 which is the counterpart of human chromosome 17). Homology in the beta subfamily ranges from 28–45% intraspecies, from 25–55% interspecies. Exemplary members include the human proteins MCP-1 (monocyte chemoattractant protein-1), LD-78 α and β, ACT-2 and RANTES and the murine proteins JE factor (the murine homologue of MCP-1), MIP-1α and β (macrophage inhibitory protein-1) and TCA-3. Human MCP-1 and murine JE factor exert several effects specifically on monocytes. Both proteins are potent chemoattractants for human monocytes in vitro and can stimulate an increase in cytosolic free calcium and the respiratory burst in monocytes. MCP-1 has been reported to activate monocyte-mediated tumoristatic activity, as well as to induce tumoricidal activity. See, e.g., Rollins, Mol. and Cell. Biol. 11:3125–31(1991) and Walter, Int. J. Cancer 49:431–35(1991). MCP-1 has been implicated as an important factor in mediating monocytic infiltration of tissues inflammatory processes such as rheumatoid arthritis and alveolitis. See, e.g., Koch, J. Clin. Invest. 90:772–79(1992) and Jones, J. Immunol. 149:2147–54(1992). The factor may also play a fundamental role in the recruitment of monocyte-macrophages into developing atherosclerotic lesions. See e.g., Nelken, J. Clin. Invest. 88:1121–27(1991), Yla-Herttuala, Proc. Nat'l. Acad. Sci. USA 88:5252–56(1991) and Cushing, Proc. Natl., Acad. Sci. USA 87:5134–38(1990).
Many of these chemokines has been molecularly cloned, heterologously expressed and purified to homogeneity. Several have had their receptors cloned. Two highly homologous receptors for the C-X-C chemokine IL-8 have been cloned and were shown to belong to the superfamily of G protein-linked receptors containing seven transmembrane-spanning domains. See Holmes, Science 253:1278–80(1991) and Murphy, Science 253:1280–83(1991). More recently, a receptor for the C-C chemokines MIP-1α and RANTES has been molecularly cloned and shown to belong to the same seven transmembrane-spanning receptor superfamily. See Gao, J. Exp. Med. 177:1421–27(1993) and Neote, Cell 72:415–25(1993). This receptor, which is believed to be involved with leukocyte activation and chemotaxis, exhibits varying affinity and signaling efficacy depending on the ligand. It binds with the highest affinity and the best signaling efficacy to human MIP-1α. To MCP-1, the receptor exhibits high binding affinity relative to RANTES and huMIP-1β but transmits signal with lower efficacy. See Neote, Id., at 421–22. Although pharmacology studies predicted the existence of a specific MCP-1 receptor, and the chemokine receptors already cloned could not account for the robust responses of monocytes to MCP-1, to date no specific receptor for MCP-1 has been reported. See Wang, J. Exp. Med. 177:699–705(1993) and Van Riper, J. Exp. Med. 177:851–856(1993). The difficulty may arise at least in part from the fact that in the chemokine family individual receptors may or may not bind multiple ligands, making functional sorting, tracking and identification impractical. It has also been speculated that the receptor members of the family may not share structural features—to account for why the MCP-1 receptor has to date eluded researchers. See Edgington, Bio/Technology II:676–81(1993).
There remains a need in the art for additional receptors to these chemokines. There also remains a need in the art for receptors specific for each of the C-C proteins, especially a receptor specific to MCP-1. Without a specific receptor to MCP-1, there is no practical way to develop assays of MCP-1 binding to its receptor. The availability of such assays provides a powerful tool for the discovery of antagonists of the MCP-1/MCP-1 receptor interaction. Such antagonists would be excellent candidates for therapeutics for the treatment of atherosclerosis in tumor growth suppression and in other diseases characterized by monocytic infiltrates such as rheumatoid arthritis and alvcolitis.